CN102395673B - Isolating method for umbilical cord blood-derived pluripotent stem cells expressing - Google Patents

Isolating method for umbilical cord blood-derived pluripotent stem cells expressing Download PDF

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CN102395673B
CN102395673B CN201080016872.7A CN201080016872A CN102395673B CN 102395673 B CN102395673 B CN 102395673B CN 201080016872 A CN201080016872 A CN 201080016872A CN 102395673 B CN102395673 B CN 102395673B
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康景宣
庐暻焕
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Seoul National University Industry Foundation
SNU R&DB Foundation
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Abstract

The present invention relates to an isolating method for umbilical cord blood-derived pluripotent stem cells which is characterized by culturing monocytes isolated from umbilical cord blood in a culture vessel containing fibronectin and then harvesting stem cells from culture, the umbilical cord blood-derived pluripotent stem cells isolated thereby, and a cell therapeutic agent containing the umbilical cord blood-derived pluripotent stem cells or differentiated cells therefrom. In addition, the present invention relates to a novel culture media for stem cells, a culture method for stem cells which is characterized by culturing and proliferating stem cells in the culture media, and a method for increasing stemness of stem cells which is characterized by sphere culture or three-dimensionalculture of stem cells.

Description

From the separation method of the omnipotent stem cell of the expression ZNF281 of Cord blood
Technical field
The present invention relates to the method for a kind of separation from omnipotent (pluripotent)/multipotency (multipotent) stem cell of Cord blood, it is characterized in that cultivating the monocyte being separated from Cord blood and obtaining in containing the culture dish of fibronectin splicing variants, then from culture, gather in the crops stem cell, thus separate from Cord blood omnipotent/multipotential stem cell; And relate to containing from Cord blood omnipotent/multipotential stem cell or the cellular therapeutic agent of cell by its differentiation.The invention still further relates to the novel culture medium of a kind of stem cell, the cultural method of a kind of stem cell, it is characterized in that, cultivate in the medium and proliferating stem cells, and a kind ofly to cultivate with the suspension ball of stem cell (sphere) or dimensional culture is the method for the stem cell properties (stemness) of the raising stem cell of feature.
Background technology
The feature of stem cell is self, differentiation occurs and is in immortal state, and stem cell has been used to the problem solving regenerative medicine and tissue substitute, and they can be used to treat various degenerative disorders, and provide deep seeing clearly to cytobiology.The adult stem cell obtained from various tissue, its magnetism is considerably beyond embryonic stem cell, because it can be derived from the ability in the source of unlimited amount, makes ethics oppose to use human embryonic cell to become unimportant as the source of cell.In addition, be separated and be compared with other the adult stem cell part that has superiority from the stem cell of Cord blood, the donor of bleeding of the umbilicus does not have injured, unlike the donor of marrow or fatty tissue.
Mescenchymal stem cell from Cord blood is successfully incorporated into various cell in vitro, comprises (people such as Sun, W., stem cell, 23:931,2005 such as neurocyte, liver cell, osteocyte; The people such as Hong SH., Biochem.Biophys.Res.Commun., 30:1153,2005; The people such as Hutson EL., organizational project (Tissue Engineering), 11:1407,2005).Also transplant in the body of reported success from the mescenchymal stem cell of Cord blood, be used for the treatment of injured, diabetes and myocardial infarction (Nonome, K. people is waited, Am, physiology magazine-stomach and intestine and liver physiology (J.Physiol.Gastrointest.Liver Physiol.), 289:1091,2005; The people such as Yoshida, S., stem cell, 23:1409,2005; The people such as Kim Bo., circulation (Circulation), 112:96,2005).Because the possibility disseminated infection is lower and cause the possibility of graft versus host disease (GVH disease) lower, transplant and be applied to children and the adult patient (people such as Claudio G.B. more and more from the mescenchymal stem cell of Cord blood, medical science year comments (Annual Review ofMedicine), 57:403,2006).Although umbilical cord blood transplantation has been accepted the treatment for some disease, particularly hemopoietic function defect relative disease (people such as Grewal, SS., blood, 103:1147,2004; The people such as Knutsen, AP., paediatrics magazine (Journal pediatrics), 142:519,2003; The people such as Ooi, J., blood, 103:489,2004; The people such as Sanz GF., Blood, 103:489,2004), still limited for the clinical application research of the mescenchymal stem cell from Cord blood.Such as, report stem cell is had to be successfully used to cure, by halves but partly cure the women of spinal cord injury and suffer from the patient (people such as Kim, SW. of thromboangiitis obliterans disease (Buerger ' s disease), stem cell, 2006; The people such as Kang, KS., cell therapy (Cytotherapy), 7:368,2005).But, the developmental mechanism of cell or they how to grow and the mechanism of breeding still is failed to understand.
Be difficult to isolate mesenchymal stem cells from Cord blood.Such as, when the method for separating of the mescenchymal stem cell from marrow is applied to Cord blood, separation rate remains on about 20%.From be separated the meat blood (flesh blood) that gathers for first 5 hours separable go out nearly 50% mescenchymal stem cell.But, when use be separated before more than 5 hours gather blood time, separation rate is down to 20% or lower, although and be separated to cell, cell proliferation is not good.
Summary of the invention
Technical problem
For completing the present invention, the present inventor has carried out deeply studying fully to the separation of the stem cell from Cord blood and a large amount of propagation, found that, when cultivating under existing at fibronectin splicing variants, be separated the monocyte hyperproliferation from Human Umbilical Cord's blood, and form cell colony, demonstrate fusiformis form, after repeatedly going down to posterity, even also maintain the characteristic of stem cell.Therefore, based on this discovery, provide a kind of effective separation from the non-hematopoiesis of Cord blood omnipotent/method of multipotential stem cell and mescenchymal stem cell and the method for extensive these stem cells of propagation.
Therefore an object of the present invention is to provide a kind of separation from Cord blood omnipotent/method of multipotential stem cell, it is included in fibronectin splicing variants exists lower culture of isolated from the monocyte of Cord blood gather in the crops stem cell from culture.
Another object of the present invention be to provide by this separation method be separated obtain from Cord blood omnipotent/multipotential stem cell.
Another object of the present invention is to provide a kind of containing the cellular therapeutic agent from the omnipotent of Cord blood or multipotential stem cell or the cell by its differentiation.
Another object of the present invention is to provide a kind of novel stem cell media.
Another object of the present invention is to provide a kind of method using described culture medium culturing stem cell.
This is that a kind of cultivation of stem cell suspension ball or dimensional culture of using is to improve the method for the stem cell properties of stem cell.
Technical scheme
According to an aspect, the present invention relates to the method be separated from the stem cell of Cord blood, it is characterized in that cultivating the monocyte being separated from Cord blood and obtaining in containing the culture dish of fibronectin splicing variants, then from culture, gather in the crops stem cell.
In one embodiment, the step gathering in the crops stem cell from culture comprises and utilizes the immunological characteristic of stem cell to be separated described stem cell.
Separating monocytic cell from Cord blood, can use typical method to realize.By Cord blood and Hetasep (cellular segregation liquid) mixing with after removing red corpuscle, use Ficoll-paque (cellular segregation liquid) separating monocytic cell.Herein, the preferred consumption of Hetasep is that every 5mL uses 0.5 ~ 2mL Hetasep.
In order to improve the output from wherein separating monocytic cell, the Cord blood used in the present invention preferably point puerperium gather immediately blood, gather after at the room temperature storage blood of 12 ~ 48 hours or store the blood of 6 ~ 72 hours after gathering at 3 ~ 5 DEG C.
From from separate stem cells the monocyte of Cord blood, it is characterized in that using fibronectin splicing variants.Term used herein " culture dish containing fibronectin splicing variants " refers to the condition that monocyte can touch with fibronectin splicing variants.Such as, fibronectin splicing variants can be laid on culture dish or with suspension ball or three-dimensional structure form and is contained in substratum by layer.In one embodiment of the invention, when with fibronectin splicing variants coating culture dish, the density of fibronectin splicing variants is 0.1 ~ 1mg/mL.
The fibronectin splicing variants used in the present invention can be derived from not limited animal, and preferred source is from the mankind.In addition, fibronectin splicing variants can be prepared by synthetic (such as, chemosynthesis, utilize peptide synthesizer synthesis etc.) or biosynthesizing is (such as, DNA recombinant technology, Fibroblast cell-culture etc.), or can comprise the blood plasma of the mankind being separated from extracellular matrix from animal and obtain.Fibronectin splicing variants can be fragment or the peptide sequence of fibronectin splicing variants, maybe can contain described fragment or peptide.
When in the culture dish that described monocyte is incubated at containing fibronectin splicing variants, adopted substratum is had no particular limits, preferably use SNU-1 or EGM-2 as the monocytic basic medium of cultivation.
SNU-1 substratum comprises following composition (table 1).
Table 1
In the present invention, described basic medium preferably supplemented with FGF-B (fibroblast growth factor), xitix, EGF (Urogastron), hydrocortisone, IGF-1 (insulin-like growth factor-i) or VEGF (vascular endothelial growth factor) and heparin, and if need optional GA-1000 (gentamicin sulphate, amphotericin B).
More preferably, described basic medium supplemented with the IGF-I (insulin-like growth factor-i) of 20% foetal calf serum (FBS), 1 ~ 40ng/ml bFGF (fibroblast growth factor), 0.1 ~ 5.0 μ g/ml xitix, 1 ~ 40ng/ml EGF (Urogastron), 0.1 ~ 1 μ g/ml hydrocortisone, 1 ~ 40ng/ml or 1 ~ 5ng/ml VEGF (vascular endothelial growth factor) and 20 ~ 25 μ g/ml heparin, and if need optionally to add GA-1000 (gentamicin sulphate, amphotericin B).
Cultivate after three days, remove the monocyte keeping suspending, and only cultivate attached cell.In the monocyte adhering to culture dish, only has stem cells hyperplasia.Be separated latter 12 ~ 20 days, can be observed the fast breeding of stem cell.In this article, preferably every couple of days changes fresh substratum.
In order to obtain from culture from Cord blood omnipotent/multipotential stem cell, FACS method (the Int.Immunol. of the flow cytometer with classification feature can be used, 10 (3): 275,1998), paramagnetic particle method or the elutriator (J.Immunol. based on mescenchymal stem cell specific antibody, 141 (8): 2797,1998).In order to obtain omnipotent/multipotential stem cell from mass propgation thing, the post that the antibody of the molecule of specific recognition cell surface expression (hereinafter referred to as " surface antigen ") is fixed thereon either individually or in combinations can be used.
Fluidic cell sorting is undertaken by droplet charge method (electrostatic droplet charging) or cell capture method.In any one method, the antibody of specific recognition surface antigen is fluorescently labeled, and measures the fluorescence intensity of the antibody-antigene binding substances of mark afterwards and is converted into electrical signal, to measure the antigen presentation amount of cell.Further, the fluorophor that use capable of being combined is different is to distinguish the cell of expressing different surfaces antigen.Fluorophor is FITC (fluorescein isothiocyanate), PE (phycoerythrin), APC (allophycocyanin), TR (texas Red), Cy3, CyChrome, Red613, Red670, three fluorescence (TRI-Color) and QuantumRed.
In the FACS method using flow cytometer, by such as centrifugal stem cell of gathering in the crops from culture, directly with antibody mediated immunity dyeing, or can rise in value in suitable substratum before with antibody mediated immunity dyeing.In order to carry out immunostaining, the primary antibodie of target cell sample with specific recognition surface antigen being mixed, and hatches 0.5 ~ 1 hour on ice.If described primary antibodie is marked by fluorophor, cleaning cell sample, and be separated with flow cytometer.If described primary antibodie is not marked by fluorophor, clean the cell sample by a process resistant, and can mix in conjunction with two of described primary antibodie are anti-with fluorescently-labeled.Then, the cell of immunostaining is hatched 0.5 ~ 1 hour again on ice, and cleans before flow cytometric sorting.
According to the present invention be separated from Cord blood omnipotent/multipotential stem cell has at least one following characteristics:
A () demonstrates positive immune characteristic to transcription factor c-myc and ZNF281;
B () adheres to the surface being coated with extracellular matrix, and within 5 ~ 30 days, form fusiformis or spherical cell colony afterwards in adhesion;
C () CPDL (accumulation population doublings level) (cumulative population doublinglevel) is 30 ~ 45;
D () demonstrates negative immune characteristic to CD14, CD31, CD34, CD45 and HLA-DR;
E () has the ability being divided into mesoderm, entoderm and ectoderm cell; And
F () secretion at least one is selected from cytokine or the chemokine of lower group: TIMP-2, TGF-β, RANTES CINC-3, EOTAXIN (chemotactic factor for eosinophils), GM-CSF, IFN-γ, IL-1b, IL-3, IL-6, IL-8, IL-10, IL12p40, IL13, IL-16, IP-10, leptin, MCP-2, MIG, MIP-3a, b-NGFm, sTNFRI and PFGF-bb.
From stem cell, express the situation of Oct-4, SOX-2, REX-1, c-myc and ZNF281, of the present invention from Cord blood omnipotent/multipotential stem cell still keeps the undifferentiated fact to be apparent.
In addition, CPDL (accumulation population doublings level) is 30 ~ 45, of the present invention from Cord blood omnipotent/multipotential stem cell is hyperproliferation.Chromosome karyotype analysis shows, cell fast breeding of the present invention, but has normal chromosome structure.
Of the present invention from Cord blood omnipotent/multipotential stem cell to CD14, CD31, CD34, CD45 and HLA-DR, be allly known as hemopoietic stem cell mark (marker) or immunological rejection correlating markings, in immunonegative.Owing to lacking hematopoiesis or immunological rejection correlating markings, the stem cell from Cord blood of the present invention can minimum vascularization and immunological rejection be transplanted, thus can effectively for heteroplastic transplantation.
Of the present invention from Cord blood omnipotent/multipotential stem cell also from mesodermal differentiation hepatoblast, can be divided into neurone and retina relevant cell from ectoderm, and becomes scleroblast, chondrocyte and adipocyte from mesodermal differentiation.Therefore, of the present invention from Cord blood omnipotent/multipotential stem cell may be used for treat various diseases.
In addition, of the present invention from Cord blood omnipotent/multipotential stem cell can secrete cytokine profiles or chemokine, comprise TIMP-2, TGF-β, RANTES CINC-3, EOTAXIN, GM-CSF, IFN-γ, IL-1b, IL-3, IL-6, IL-8, IL-10, IL12p40, IL13, IL-16, IP-10, leptin, MCP-2, MIG, MIP-3a, b-NGFm, sTNFRI and PFGF-bb.The present invention with these cytokines of secretion or chemokine ability from Cord blood omnipotent/multipotential stem cell can be used for treating various disease.
Of the present invention from Cord blood omnipotent/new property of multipotential stem cell comes from them and has these features.
As mentioned above, of the present invention from Cord blood omnipotent/multipotential stem cell can be divided into various types of cell, comprise scleroblast, chondrocyte, adipocyte, liver cell and neurone, thus it finds the application in various corresponding physics.Therefore, the invention provides a kind of containing of the present invention from Cord blood omnipotent/multipotential stem cell or the cellular therapeutic agent of cell by its differentiation.Cellular therapeutic agent of the present invention can be used for treating various disease, such as, nervous system disorders (as nerve degenerative diseases), osteoarthritis (as osteoarthritis, rheumatoid arthritis), bone-loss (as osteoporosis), hepatic diseases (as liver cirrhosis) and cardiovascular disorder is comprised.
Preferably, cellular therapeutic agent of the present invention comprises the thinner that cell could be protected and maintain at least one.Damping fluid such as physiological saline, PBS (phosphate buffered saline buffer), HBSS (hanks' balanced salt solution) (Hank ' s balanced salt solution) and blood plasma or serum composition can be used as thinner.
According to its another aspect, the present invention relates to the substratum cultivating novel stem cells.Described substratum is based on EGM-2 or SNU-1 substratum, and supplemented with 20% foetal calf serum (FBS), 1 ~ 40ng/ml bFGF (fibroblast growth factor), 0.1 ~ 5.0 μ g/ml xitix, 1 ~ 40ng/ml EGF (Urogastron), the IGF-I (insulin-like growth factor-i) of 0.1 ~ 1 μ g/ml hydrocortisone, 1 ~ 40ng/ml or 1 ~ 5ng/ml VEGF (vascular endothelial growth factor) and 20 ~ 25 μ g/ml heparin, and if need optional GA-1000 (gentamicin sulphate, amphotericin B).
The substratum of described culturing stem cells is novel culture medium, be applied to being separated of the present invention from Cord blood omnipotent/method of multipotential stem cell in.In addition, substratum of the present invention is applicable to breed all adult stem cells comprising stem cell from Cord blood, and is applicable to the cultivation of adult stem cell.
According to its another aspect, the present invention relates to a kind of method of culturing stem cells, be included in substratum of the present invention and cultivate and proliferating stem cells.Preferably, described stem cell can be adult stem cell.
In one embodiment, the substratum of culturing stem cells of the present invention can be used for cultivate of the present invention from Cord blood omnipotent/multipotential stem cell.When cultivating in substratum of the present invention, of the present invention from Cord blood omnipotent/multipotential stem cell can preferably go down to posterity after 3 ~ 5 days at formation spindle cell colony.Preferred cultivation is at 5%CO 2under condition, and can 5 ~ 30 days be carried out, but the invention is not restricted to these.
According to its another aspect, the present invention relates to a kind of method improving the stem cell properties of stem cell, it is characterized in that using the cultivation of the suspension ball of stem cell or dimensional culture to cultivate stem cell.For dimensional culture, preferably use MEF (mouse embryo fibroblasts).In addition, stem cell can be preferably adult stem cell.
Term used herein " raising stem cell properties " refers to and forms embryonic stem cell-like cell colony or express transcription factor as Oct4, Sox2 etc. with higher level.
Beneficial effect
As detailed above, when cultivating under fibronectin splicing variants existent condition, of the present invention from Human Umbilical Cord's blood omnipotent/multipotential stem cell, compared with traditional adult stem cell, can to breed actively for a long time and undifferentiated.In addition, have and be divided into various cell, as the ability of chondrocyte, scleroblast and adipocyte the present invention from Cord blood omnipotent/multipotential stem cell can effectively for conventional difficult and complicated cases, and nervous system disorders, cardiovascular disorder, disease of skeletal system treatment.
Accompanying drawing explanation
The photo of Fig. 1 shows to be isolated monocyte and cultivates the cell colony that 14,15,16,17 and 18 days (being respectively A, B, C, D and E) and cell formed after 3 times go down to posterity (F) from human cord blood;
Fig. 2 is the figure that accumulation Growth of Cells was drawn the time;
Fig. 3 show from Cord blood omnipotent/multipotential stem cell cultivated very long one section time after date karyotyping result.
The fluidic cell figure of Fig. 4 show the present invention from Cord blood omnipotent/multipotential stem cell on the expression pattern of various marks.
Fig. 5 show adopt the present invention of cells were tested by flow cytometry from Cord blood omnipotent/multipotential stem cell on various do not break up mark expression pattern and immunostainings (A: the fluidic cell figure expressing Oct4 cell, B: the Oct4 image of immunostaining, the superimposed images of C: the nuclear stain image of expressing Oct4 cell, D:Oct4 expression and nuclear staining).
Fig. 6 shows the expression (upper figure) adopting ZNF281 in terra-1, hUCB-MSC, AD-MSC and AM after the going down to posterity through 3 ~ 9 times of facs analysis, and the expression (figure below) of ZNF281 in hUCB-MSC.
Fig. 7 is shown and is measured by RT-PCR, all for maintain do not break up necessary ZNF281, Oct4, Sox2, c-myc and Rex-1 of the present invention from Human Umbilical Cord blood omnipotent/multipotential stem cell in expression.
The photo of Fig. 8 show from Human Umbilical Cord's blood omnipotent/pluripotent stem cell differentiation is scleroblast, adipocyte, chondrocyte and neurone (A: the control of non-Osteoinductive differentiation after alizarin red S dyeing, B: scleroblast differentiation-inducing after alizarin red S dyeing, C: the control of non-adipogenic induction differentiation after oil red O stain, D: adipocyte differentiation-inducing after oil red O stain, E: chondrocyte differentiation-inducing after Toluidine blue staining, F: the particle of differentiation-inducing chondrocyte, G: the cell after neural cellular differentiation with the immunostaining of neural marker Tuj-1 and MAP2.
Fig. 9 is shown and to be measured by RT-PCR, through be induced to differentiate into scleroblast after adipocyte from Cord blood omnipotent/(PPAR-γ with FABP-4 is into the mark of fat differentiation, and 1 collagen type is the mark of Osteoblast Differentiation for the rna level of multipotential stem cell.PPAR-γ: the receptor y of peroxisome proliferation activation, FABP-4: FABP4, GAPDH:3-phosphoglyceraldehy-de dehydrogenase).
Figure 10 show of the present invention from Cord blood omnipotent/multipotential stem cell in the expression mould of retina associated protein.
Figure 11 is shown and to be analyzed by antibody chip, from Cord blood omnipotent/the various cytokine (A:hUCB-MSC1 that secrete in substratum of multipotential stem cell, B:hUCB-MSC2, C:hUCB-MSC3, D: antibody is arranged, POS: positive control, NEG: negative control, GCSF: granulocyte colony-stimulating factor, GM-CSF: granulocyte Granulocyte microphage colony stimulating factor, ICAM-1: adhesion molecule in born of the same parents, IFN-γ: interferon-γ, IL: interleukin-, MCP: MCP, M-CSF: Granulocyte microphage colony stimulating factor, MIG: the monokine of interferon-gamma induction, MIP: macrophage inflammatory protein, RANTES: the factor regulating normal T-cell expression and secretion, TGF-β: transforming growth factor-beta, TNF: tumour necrosis factor, sTNFR: soluble tumor necrosis factor receptor, PDGF-BB: PDGF-BB, TIMP2: Tissue inhibitor-of metalloproteinase-2).
The photo of Figure 12 and 13 show adopt suspension ball culture method from Cord blood omnipotent/the dimensional culture thing of multipotential stem cell and STO cell, and the expression pattern of the transcription factor Oct4 to be measured by RT-PCR and Sox2 (Figure 12: adopt suspension ball culture method from Cord blood omnipotent/the dimensional culture thing of multipotential stem cell, Figure 13: based on STO cell from Cord blood omnipotent/the dimensional culture thing of multipotential stem cell)
Embodiment
Understand the present invention better by the following examples, it is for illustration of object, but is not used for limiting the present invention.
embodiment 1: from Cord blood omnipotent/cultivation of multipotential stem cell, propagation and karyotyping
Through mother's informed consent, gather mature Cord blood (UCB) sample (n=20) immediately in a point puerperium.Mature Cord blood is mixed (Stemcell Technologies Inc. (CA), Vancouver, BC) with HetaSep to remove red corpuscle.Then, typical Ficoll density gradient centrifugation is adopted to obtain monocyte.By monocyte with 1 × 10 5 ~ 8the density of individual cells/well is inoculated in and is coated with in 6 orifice plates of 0.1mg/ml ~ 1mg/ml fibronectin splicing variants, and is held in SNU-1 or EGM-2 (Lonza) supplemented with the EGM-2 SingleQuots containing 20% foetal calf serum.The EGM-2 of described SingleQuots comprises heparin, xitix, recombinant human epidermal growth factor, hydrocortisone, vascular endothelial growth factor, recombinant human fibroblast somatomedin, human insulin-like growth factor and GA-1000.Remove the monocyte that cultivation is adherent not yet after 3 days, every couple of days changes fresh substratum simultaneously.
Observe attached cell and form cell colony, at 5%CO 2condition under cultivate after 5 ~ 30 days, demonstrate fusiformis form (Figure 1A).Once formation colony, cell colony fast breeding.To form after colony 3 ~ 7 days with 0.125% trypsinase-EDTA suspension cell, and transfer to fresh plate relaying continuation of insurance and hold cell (Figure 1B, 1C, 1D, 1E and 1F).The photo of Fig. 1 shows the cultivation progress of cell.As shown in Figure 1, passing in time, cell colony becomes large gradually.In addition, even if after going down to posterity, the form of cell is still consistent (Fig. 1, from Cord blood omnipotent/separation of multipotential stem cell and propagation.After monocyte cultivates 14 days (A), 15 days (B), 16 days (D), 17 days (D) and 18 days (E), and the cell colony formed after 3 times go down to posterity (F).
While continuation culturing cell, by measure that CPDL (accumulation population doublings level) (cumulative population doubling level) come that analytical separation obtains from Cord blood omnipotent/ability of the proliferation of pluripotent stem cells (people such as Cristofalo, Proc.Natl.Acad.Sci., USA 95,1998).Cell carries out binary fissiparity.Therefore, the speed of growth of cell can become time required for two cells according to a cell fission, is called that the doubling time is determined.CPDL is 10 expression cell fission 10 times, and result causes a cell proliferation for about 1000 cells.One of greatest problem of traditional stem cell from Cord blood is, compared with the mescenchymal stem cell from fatty tissue or marrow, they have significantly low multiplication capacity.Because clinical application needs a large amount of stem cells, the multiplication capacity of stem cell is very important.Be determined as follows.First, isolate from different Cord blood samples three kinds from Cord blood omnipotent/multipotential stem cell, and with 2 × 10 5the density of individual cell is inoculated in 100 plates, regularly goes down to posterity at three to four days, uses cell counter to cell counting simultaneously.Until count cell when cell stops growing.Based on cell counting, obtain CPDL value according to mathematical formula 1 below.
[mathematical formula 1]
N h/ N i=2 xor [log (N h)-log (N i)]/log (2)=X
Wherein, N irepresent the quantity of cell in initial medium, N hrepresent the quantity of cell after going down to posterity under state of saturation.
While continuing to keep cell, calculate CPDL value.HUCB-EPC (endothelial progenitor cells from human cord blood) is in contrast for comparing.Result as shown in Figure 2.From in the table of Fig. 2, hUCB-EPC has CPDL about 20 after two months, and observe whole three different samples from Cord blood omnipotent/CPDL of multipotential stem cell is 40 ~ 45.These numerical value show, cell can grow and reach 10 in a colony theoretically 12the quantity of individual cell.
Generally, may there is canceration in the cell with stronger multiplication capacity, occur explosive growth.Once generation canceration, cell may be ignored the various adjustment signal in body and breed, and thus can not be used as cellular therapeutic agent and inconsistent with research purpose.Therefore, must determine the present invention be separated obtain from Cord blood omnipotent/whether the karyomit(e) of multipotential stem cell is normal.In this article, chromosome karyotype analysis is carried out to detect the karyomit(e) of cell.As can be seen from Figure 3, even after 10 times go down to posterity, find that cell has normal chromosome structure.
embodiment 2: from Cord blood omnipotent/surface antigen analysis of multipotential stem cell
Flow cytometry is adopted to analyze the cell characteristics be suspended in substratum.In order to determine the phenotype of cell-surface antigens, after going down to posterity through 3 ~ 4 times with in conjunction with fluorescein isothiocyanate (FITC)-or in conjunction with phycoerythrin (PE)-antibody harvested cell is dyeed, and adopt FACSAria (Becton Dickinson, NY) to analyze.
Be used for analyzing the present invention be separated obtain from Cord blood omnipotent/surface antigen of multipotential stem cell (part omnipotent/multipotential stem cell) characteristic comprises CD10 (T cell mark), CD14 (monocyte mark), CD24 (epithelial cell mark), CD29 (monocyte mark), CD31 (endothelial cell marker), CD34 (hemopoietic stem cell mark), CD44 (mescenchymal stem cell mark), CD45 (non-hematopoietic stem cell mark), CD51/61 (osteoclast mark), CD73 (mescenchymal stem cell mark), CD90 (mescenchymal stem cell mark), CD105 (mescenchymal stem cell mark), CD133 (hemopoietic stem cell mark) and HLA-DR (immunological rejection correlating markings), and adopt flow cytometer analysis.Result is summarized in table 2 and is illustrated in Fig. 4.
Table 2
From Cord blood omnipotent/multipotential stem cell on T cell differentiation antigen Positive stained cells/total cell (%)
CD10 0.2±0.1
CD14 2.0±1.0
CD24 68.7±2.9
CD29 100±0.0
CD31 0.4±0.7
CD34 3.5±3.4
CD44 100±0.1
CD45 0.0±0.1
CD51/61 6.4±10.8
CD73 99.6±0.5
CD90 99.7±0.3
CD105 99.5±0.8
CD133 0.1±0.1
HLA-DR 2.0±3.2
the expression pattern of embodiment 3:ZNF281 and from Cord blood omnipotent/multipotential stem cell in core transcription factor
ZNF281 (zinc finger protein 28 1) is one of core transcription factor of ESC (people such as Wang J, (2006) nature 444,364-368).ZNF281, also known as ZBP-99, comprise four Kruppel type zinc and refer to, it has the amino acid sequence similarity of 91% jointly, and with find in ZBP-89 have 79% sequence homology.In addition, there is the aminoacid sequence of high conservative in the carboxy terminal fragment of these two genes.The albumen of the open reading frame coding 99kDa of the ZNF281 cDNA of prediction.EMSA (being called electrophoretic migration) shows, ZNF281 protein-specific is attached to the promoter element (people such as Law DJ of being rich in GC of gastrin and ornithine decarboxylase gene, (1999) Biochem Biophys Res Commun 262,113-120; The people such as Lisowsky T, (1999) FEBS Lett 453,369-374).Adopt mass spectrum multidimensional protein identification techniques and tandem affinity purification, identify that ZNF281 is a kind of c-MYC gene-correlation albumen people such as (, (2007) cell cycle (Cell Cycle) 6,205-217) Koch HB.
Known Oct3/4 gene, as POU family transcription factor, do not exist in differentiated tissue, but in the undifferentiated stem cell with higher multiplication capacity, express (the people such as Tai M-H. especially, carcinogenesis (Carcinogenesis) 26:495,2005; The people such as Tondreau T., stem cell, 23:1105,2005).Generally speaking, therefore Oct3/4 is used as the mark of embryonic stem cell, also can as the undifferentiated mark of instruction.Dye as the mark cell cluster of stem cell properties with Oct4.As a result, find that many cells have the dyeing of Oct4 around nucleus.Flow cytometry also shows the expression of Oct4 in cell.
In order to the intracellular protein that dyes, with 4% formaldehyde in 4 DEG C of fixed cells that spend the night, permeate 10min with 0.1%TritonX-100 (Sigma-Aldrich).Slide and plate and anti-human Oct4 mouse primary antibodie (1: 200) are hatched one hour, with PBS (phosphate buffered saline buffer; Gibco) rinse, resist (Invitrogen) immunostaining one hour with the sheep anti-mouse igg two in conjunction with Alexa594, meanwhile, nucleus DAPI redyes.
As illustrated in Figures 5 and 6, find many from Cord blood omnipotent/multipotential stem cell expresses Oct4 (Fig. 5 A: the fluidic cell figure expressing Oct4 cell, B: the Oct4 image of immunostaining, C: the nuclear stain image of expressing Oct4 cell, the superimposed images of D:Oct4 expression and nuclear staining).
Gene, as Oct4 gene, has close association with the stem cell properties of stem cell.In fact, attempt by making cell process LAN gene as Oct4, Sox2 etc. recently, adult stem cell is induced into there is versatility (the pluripotency) (people such as Takahashi similar with embryonic stem cell, cell, 131 (5), 861-872,2007).Therefore, the expression of these genes in stem cell, stem cell is remained in undifferentiated state and do not lose stem cell properties is very important.Therefore, in order to determine ZNF281, Oct-4 and Sox2 the present invention be separated obtain from Cord blood omnipotent/multipotential stem cell in expression pattern, carry out RT-PCR.
For this reason, primer in table 3 is constructed for RT-PCR.
Table 3
As shown in Figure 7, Oct-4, Sox2, c-myc, ZNF281 and REX-1 are expressed, and this shows that the stem cell from Cord blood of the present invention has versatility.
embodiment 4: from Cord blood omnipotent/pluripotent stem cell differentiation is scleroblast
Cell is induced to differentiate into scleroblast.For this reason, make cell adhesion in cultivation plate and hatch to about 70 ~ 80% cell confluency (confluency).Then substratum is replaced with the substratum of inducing osteoblast differentiation.The substratum of shown inducing osteoblast differentiation obtains by adding 10% foetal calf serum, 10mM beta-glycerophosphate (Sigma-Aldrich), 0.1 μM of dexamethasone (Sigma-Aldrich) and 50 μMs of ascorbate salts (Sigma-Aldrich) in low dextrose DMEM.Within every three days, replace with fresh substratum, differentiation-inducing about two weeks.
After two weeks, through the mineralising of alizarin red S staining examine calcium to the effect of Osteoblast Differentiation.Be performed as follows dyeing.After discarding substratum, cell distilled water wash twice, and one hour is fixed in 70% cold ethanol.Then, then use distilled water wash cell twice, and at room temperature dye 10 minutes with 40mM alizarin red S, then with distillation washing five times.
As shown in figs. 8 a and 8b, without in differentiation situation in Fig. 8 A, calcium is not detected with alizarin red S, and in the fig. 8b, when cell is induced to differentiate into scleroblast, calcium occurs red, this show described from Cord blood omnipotent/pluripotent stem cell differentiation is the scleroblast of release calcium.
embodiment 5: from Cord blood omnipotent/pluripotent stem cell differentiation lipoblast
Cell is induced to differentiate into adipocyte.For this reason, make cell adhesion in cultivation plate and hatch to about 70 ~ 80% cell confluency.Then substratum is replaced with Adipocyte Differentiation substratum.Described Adipocyte Differentiation substratum by adding 10% foetal calf serum, 1 μM of dexamethasone, 10 μ g/ml Regular Insulin (Sigma-Aldrich), 0.5mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich) and 0.2mM indomethacin (Sigma-Aldrich) and obtain in low dextrose DMEM.Within every three days, replace described division culture medium with fresh substratum, differentiation-inducing about 2 ~ 3 weeks.
After 2 ~ 3 weeks, detect Adipocyte Differentiation through oil red O stain.In order to dye, discard substratum, and in PBS washed cell, and in 10% formalin, hatch 5min under room temperature.Formalin is replaced, then fixed cell at least one hour under room temperature with isopyknic fresh formaldehyde.After discarding formalin, with 60% Virahol cleaning cell.Before cell uses oil red O stain 10min under room temperature, evaporate alcohol completely.After removing dyestuff, in distilled water, clean cell immediately.
After oil red O process, adipocyte occurs red, incarnadines because fat drips by it.As is shown in fig 8 c and fig 8d, fat-free cytodifferentiation in Fig. 8 C, does not detect that fat drips or red appearance; And in Fig. 8 D, when cell is induced to differentiate into adipocyte, observing cell contains much fat and drip, thus become red.
embodiment 6: from Cord blood omnipotent/pluripotent stem cell differentiation chondroblast
In order to Cell differentiation inducing activity chondroblast, process cell three weeks with the chondroblast division culture medium (PT-3003) purchased from Lonza containing rTGF β-3, twice substratum is replaced to fresh substratum weekly.Survey a chondroblast weekly.
In order to be detected as Chondrocyte Differentiation, carry out Toluidine blue staining.Fix 10 hours with 4% formaldehyde, then fix 10 hours again with picric acid.After carrying out cell section, with Toluidine blue staining cell 3min, and use haematoxylin redyeing 3sec.
Chondroblast particle does not destroy, and keeps constant form, and when presenting blueness with during Toluidine blue staining.As shown in Fig. 8 E and 8F, observe the cell experienced to chondroblast differentiation and be dyed to blueness, and keep their form.
embodiment 7: from Cord blood omnipotent/pluripotent stem cell differentiation is scleroblast and adipocyte the change of rear gene expression dose
In experience atomization, observe the noticeable change of stem cell gene expression pattern.As a rule, along with stem cell is to Adipocyte Differentiation, PPAR-γ (receptor y of peroxisome proliferation activation) or the expression level of FABP4 (FABP4) raise, and along with stem cell to osteoblast differentiation, the expression level of 1 Collagen Type VI raises (people such as Mat hews, J Am Acad Dermatol, 56 (3), 472-492,2007; The people such as Cho, J. cellular biochemistry (Cell.Biochem.), 96,533-542,2 005).Therefore, after differentiation-inducing, the gene expression dose of expressing in specific cell type shows whether stem cell is divided into the cell of this particular type indirectly.Carry out following experiment.
Be divided into scleroblast and after adipocyte two to three week, extract cell total rna with Trizol (Invitrogen).Then adopt AccuPower RT Premix (Bioneer) to synthesize cDNA uses Maxime PCR PreMix test kit (Intronbio) to carry out PCR detection.
In order to carry out PCR detection, design the primer below shown in table 4.
Table 4
Obviously can find out from the data Fig. 9, when cell is induced to differentiate into adipocyte, the mark of Adipocyte Differentiation, the expression level of PPAR-γ and FABP4 improves greatly.Equally, the mark of osteoblast differentiation, the expression level of 1 Collagen Type VI greatly improves in the cell of experience differentiation.Meanwhile, in the cell experiencing and do not experience differentiation, internal reference (loading control) GAPDH of par detected, support the reasonableness of experiment.
embodiment 8: from Cord blood omnipotent/pluripotent stem cell differentiation is neurone
Cell is induced to differentiate into neurone.First, by cell preincubate 24 hours in the DMEM supplemented with 5%FBS and 10ng/ml bFGF (Prostatropin).After this, cell processes and breaks up with inducing neural for 24 hours in the DMEM containing 1%DMSO, 100 μMs of BHA, 0.5mM VPA, 10mM KCl and 10ng/ml NGF and B27.In order to detect Neural Differentiation, with 4% paraformaldehyde fixed cell and immunostaining neural marker Tuj-1, MAP-2, GFAP and neurofilament protein-160.As a result, observe the expression (Fig. 8 G) of four marks.
embodiment 9: from the expression of retinal specific albumen in the mescenchymal stem cell of Cord blood
With Immunofluorescence test from Cord blood omnipotent/the retina correlation properties of multipotential stem cell.
Detect the expression pattern of retinal specific albumen in cell.
The photo of Figure 10 shows the expression pattern of the retinal specific albumen by immunofluorescence assay.The expression pattern of PAX6 and Hu albumen is shown in Figure 10 A.Known PAX6 is retinal progenitor cells mark, and Hu protein-specific is expressed in as in the retinal ganglial cells of retina composition and amacrine cell.Their expression is can't detect in normal culture.
In fig. 1 ob, opsin and Visual purple is detected.Opsin is specific expressed in cone cell, and Visual purple is special for rod photoreceptor cell.In normal culture, can't detect opsin, and observe the expression of Visual purple.
Figure 10 C is depicted as the expression pattern of CRX and recoverin.Known CRX is floodlight susceptor mark, and recoverin is sight sensor mark.In normal culture, can't detect these marks (magnification × 400, scale=50 μm).
embodiment 10: from Cord blood omnipotent/analysis of cytokine of multipotential stem cell release
The ability of stem-cell therapy depends on two factors to a great extent: the first, and stem cell is directly divided into damaged cell; The second, secrete various ability of bringing out cytokine or the somatomedin actively changed, thus cause the result for the treatment of to both depositing cell.In general, the various cytokine of known stem cell secretion or somatomedin, demonstrate so-called paracrine action (people such as Kim, cytokine, 2005).In order to detect the present invention be separated obtain from Cord blood omnipotent/the secretion pattern of multipotential stem cell, adopt Human cytokine's antibody chip (RaybioTech.Norc ross, USA).
First, in the substratum not containing FBS and feed supplement, stabilized cell 24 hours, then got the substratum of 1mL every 2 hours.Each culture sample gets 100 μ L formation for the pond of protein level quantitative analysis, then detects on chip.
As shown in figure 11, find whole three different samples from Cord blood omnipotent/multipotential stem cell all secretes IL-8 and TIMP-2.In addition, also observe the various cytokine of secretion, comprise TGF-β, RANTES, CINC-3, EOTAXIN, GM-CSF, IFN-γ, IL-1b, IL-3, IL-6, IL-10, IL12p40, IL13, IL-16, IP-10, leptin, MCP-2, MIG, MIP-3a, b-NGFm, sTNFRI and PFGF-bb (Figure 11 is the Microarray results of hUCB-MSC1 (A), hUCB-MSC2 (B) and hUCB-MSC3 (C) and antibody arrangement (D)).
embodiment 11: from Cord blood omnipotent/dimensional culture of multipotential stem cell
In general, adult stem cell grows and forms monolayer cell in culture dish.But experiment shows, when adult stem cell is not with two-dimensional state, but during to grow as suspension ball with three-dimensional state, the stem cell properties of adult stem cell strengthens.For this reason, first, with 0.7% agarose, culture dish is coated into the thickness of 5mm or more, makes cell cannot adhere to bottom, but form suspension ball.With lower than 2000 cell/cm 2density inoculating cell to prevent the adhesion between individual cells.Thus use 40 μm of strainers to be separated from unicellular by the suspension ball of formation.As can be seen from Figure 12, there is not necrocytosis in stem cell, but form suspension ball, when cultivating in suspension ball culture system, maintains the characteristic of stem cell.As shown in Figure 12 A-D, observe embryo's mark, as OCT4, SOX2 etc. keep the cell of individual layers higher compared with those being held in the expression level in suspension ball cultured cells.
Embryonic stem cell is cultivated usually on the cellular layer of mouse embryo fibroblasts, because chemokine Tathagata is from the LIF of mouse embryo fibroblasts, is maintaining the form of ES cell and is preventing from having played vital role in the differentiation of ES cell.When on the cellular layer being incubated at mouse embryo fibroblasts, from Cord blood omnipotent/multipotential stem cell unlike adult stem cell grow into flat-section, but form three-dimensional cell colony.After suppress its propagation with 0.1mg/ml ametycin, with 2 × 10 5the plate that STO cell is inoculated in coating 0.1% gelatin by the density of individual cell/ml is cultivated 24 hours, then by from Cord blood omnipotent/multipotential stem cell is inoculated on STO cellular layer.As shown in figure 13, observe As time goes on, cell is bred with the pattern being similar to embryonic stem cell on STO cell.
Industrial applicibility
When cultivating under fibronectin splicing variants existent condition, compared with traditional adult stem cell, of the present invention from human cord blood omnipotent/multipotential stem cell breeds longer for some time actively and do not break up.In addition, the present invention is omnipotent/multipotential stem cell has and be divided into various cell, as chondrocyte, scleroblast and adipocyte ability, can be effectively applied to conventional difficult and complicated cases, and nervous system disorders, cardiovascular disorder, disease of skeletal system treatment.

Claims (19)

1. one kind is separated from the expression ZNF281 of Cord blood from Cord blood, Sox-2, Oct-4, the method of the stem cell of c-myc and Rex-1, it is included in the monocyte cultivated in the culture dish containing fibronectin splicing variants and be separated from Cord blood and obtain, then from culture, gather in the crops the step of the stem cell of cultivation, wherein said monocyte be incubated at supplemented with 20% or be less than 20% foetal calf serum (FBS), 1 ~ 40ng/ml bFGF, 0.1 ~ 5.0 μ g/ml xitix, 1 ~ 40ng/mlEGF, 0.1 ~ 1 μ g/ml hydrocortisone, in the substratum of the IGF-I of 1 ~ 40ng/ml or 1 ~ 5ng/ml VEGF and 20 ~ 25 μ g/ml heparin.
2. method according to claim 1, wherein monocytic separation is by mixing Cord blood and Hetasep to remove red corpuscle, then using Ficoll-paque.
3. prepare a method for the stem cell of expression ZNF281, Sox-2, Oct-4, c-myc and the Rex-1 from Cord blood, it comprises the cell isolated from Cord blood and ZNF281, Sox-2, Oct-4, c-myc and Rex-1 are demonstrated to positive immune characteristic.
4. method according to claim 1, wherein said stem cell results cultivate the cell colony formed afterwards for 5 ~ 30 days certainly.
5. method according to claim 1, wherein said fibronectin splicing variants layer is laid on culture dish or with suspension ball or three-dimensional structure form and is contained in substratum.
6. method according to claim 5, wherein when culture dish fibronectin splicing variants is coated with, the density of contained fibronectin splicing variants is 0.1 ~ 1mg/mL.
7. method according to claim 1, wherein said fibronectin splicing variants is derived from animal.
8. method according to claim 7, wherein said animal is the mankind.
9. method according to claim 3, it comprises cultivates in the mode of suspension ball cultivation and dimensional culture cell ZNF281, Sox-2, Oct-4, c-myc and Rex-1 being demonstrated to positive immune characteristic further.
10. method according to claim 1, the step wherein gathering in the crops the stem cell of cultivation from culture comprises and utilizes the immunological characteristic of stem cell to be separated described stem cell.
The 11. couples of transcription factor ZNF281, Sox-2, Oct-4, c-myc and Rex-1 demonstrate the stem cell from Cord blood of positive immune characteristic.
12. stem cells from Cord blood according to claim 11, it is omnipotent or multipotential stem cell.
13. stem cells from Cord blood according to claim 11, it adopts method according to claim 1 to be separated and obtains, and has the following characteristic of at least one:
A () demonstrates positive immune characteristic to transcription factor c-myc, Sox-2, Rex-1, Oct-4 and ZNF281;
B () adheres to the surface that is coated with extracellular matrix and within 5 ~ 30 days, forms fusiformis or spherical cell colony afterwards in adhesion;
C () cellular accumulation multiplier stage is 30 ~ 45;
D () demonstrates negative immune characteristic to CD14, CD31, CD34, CD45 and HLA-DR;
E () has the ability being divided into mesoderm, entoderm and ectoderm cell; And
F () secretion at least one is selected from cytokine or the chemokine of lower group: TIMP-2, TGF-β, RANTES CINC-3, EOTAXIN, GM-CSF, IFN-γ, IL-1b, IL-3, IL-6, IL-8, IL-10, IL12p40, IL13, IL-16, IP-10, leptin, MCP-2, MIG, MIP-3a, b-NGFm, sTNFRI and PFGF-bb.
14. 1 kinds of cellular therapeutic agent, it comprises the stem cell from Cord blood according to claim 11 or the cell by its differentiation.
15. for the purposes of the substratum of the stem cell of expression ZNF281, Sox-2, Oct-4, c-myc and the Rex-1 from Cord blood in method described in claim 1, described substratum comprise supplemented with 20% or be less than 20% foetal calf serum (FBS), 1 ~ 40ng/ml bFGF, 0.1 ~ 5.0 μ g/ml xitix, 1 ~ 40ng/ml EGF, EGM-2 or the SNU-1 substratum of the IGF-I of 0.1 ~ 1 μ g/ml hydrocortisone, 1 ~ 40ng/ml or 1 ~ 5ng/ml VEGF and 20 ~ 25 μ g/ml heparin.
The method of the stem cell of 16. 1 kinds of culture expression ZNF281, Sox-2, Oct-4, c-myc and Rex-1, comprise and use following substratum, described substratum comprise supplemented with 20% or be less than 20% foetal calf serum (FBS), 1 ~ 40ng/ml bFGF, 0.1 ~ 5.0 μ g/ml xitix, 1 ~ 40ng/ml EGF, 0.1 ~ 1 μ g/ml hydrocortisone, the IGF-I of 1 ~ 40ng/ml or EGM-2 or the SNU-1 substratum of 1 ~ 5ng/ml VEGF and 20 ~ 25 μ g/ml heparin.
17. methods according to claim 1, also comprise use stem cell suspension ball cultivate or dimensional culture to improve the dryness of stem cell.
18. methods according to claim 17, wherein mouse embryo fibroblasts is used for described dimensional culture.
19. methods according to claim 18, wherein said stem cell is adult stem cell.
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